Multiprobe laser reflectometry in imaging and characterization of biological tissues

Laser backscattered radiation from human forearm and foot were measured by multi-probe reflectometer, which consisted of one input probe and three output probes placed at distances of 2, 4 and 6 mm from the input probe. The normalized backscattered intensity (NBI) signals from the tissue surface, measured by the output probes, after digitization, were used to reconstruct the reflectance images of tissues in various layers below the skin surface. From NBI profiles measured at various locations of the tissues on the forearm the corresponding optical parameters, the scattering (mu(s)) and absorption (mu(a)) coefficients and the anisotropy parameter g, by matching these with profiles as simulated by Monte Carlo procedure were determined. From these data the optical parametric images of forearm were reconstructed which show the variation of these parameters at various locations. Similarly, the NBI data were collected from the foot sole region of healthy and diabetes subjects and their images reconstructed. These images showed the variation in the NBI in the diabetic foot sole compared to that of healthy subject, indicating the tissue structural changes. These procedures could be useful for diagnostic and therapeutic applications of lasers.     

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.     

Optical Shading Induces an In‐Plane Potential Gradient in a Semiartificial Photosynthetic System Bringing Photoelectric Synergy

Semiartificial photosynthetic systems have opened up new avenues for harvesting solar energy using natural photosynthetic materials in combination with synthetic components. This work reports a new, semiartificial system for solar energy conversion that synergistically combines photoreactions in a purple bacterial photosynthetic membrane with those in three types of transition metal–semiconductor Schottky junctions. A transparent film of a common transition metal interfaced with an n‐doped silicon semiconductor exhibits an in‐plane potential gradient when a light‐penetration variance is established on its surface by optical shading of photoabsorbing photosynthetic membranes. The in‐plane potential gradients (0.08–0.3 V) enable a directional charge transport between the synthetic and natural photoelectric systems, which is further enhanced in a device setting by a biocompatible thixotropic gel electrolyte that permeates the membrane multilayer, facilitating a strong and steady photoelectric current as high as 1.3 mA cm^?2, the highest achieved so far with any anoxygenic photosynthetic system.     

Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration

Summary Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. Pusa Kalyani. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and -glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450–625 from the cut surface. Such shoots were negative for GUS activity.     

Monitoring of low concentrations of glucose in fermentation broth

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.     

Flow injection analysis of intracellular β-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification

A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10^-7 mol/L.     

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline^reg that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.     

A low temperature microbial biosensor using immobilised psychrophilic bacteria

A psychrophilic bacteria, Deinococcus radiodurans, was used to construct a biosensor to be used in a flow injection system. The transducer used was an O₂ electrode. The response of this cell-based electrode was studied towards a number of sugars. The temperature dependence of the electrode response correlated well with the behavior of the cells. Thus, the optimum temperature for measurement of glucose (0.55 mM) was about +5°C. Since the organism used is psychrophilic, a response time at this low temperature is similar to what is achieved with mesophilic organisms at room temperature. This is the first biosensor constructed using a psychrophilic microorganism.     

Association of the Hydrolytic Enzyme Chitinase against Rhizoctonia solani in Rhizobacteria-treated Rice Plants

Twenty‐two strains of Pseudomonas fluorescens isolated from the rhizosphere soil of nine plant species were screened in vitro for their inhibitory effect on the mycelial growth of the rice sheath blight fungus, Rhizoctonia solani. Of the 22 strains, two promising strains (Pf1 and FP7) were assessed for their effect on seedling vigour and their ability to promote growth in vitro of four cultivars of rice. Both bacterial strains induced systemic resistance in rice cv. IR 50, which is susceptible to sheath blight. After inoculation of the sheaths with the pathogen, Pseudomonas‐treated plants showed an increase in chitinase activity significantly higher than that of untreated control plants. A twofold increase in chitinase activity occurred 2 days after inoculation of plants with the pathogen. Western blot analysis of chitinase indicated the expression of 28 and 38 kDa proteins in rice sheaths against R. solani. Increased induction of the pathogenesis‐related chitinase isoform in Pseudomonas‐treated rice in response to R. solani infection indicates that the induced chitinase has a definite role in suppressing disease development.     

Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.